U.S. patent number 7,098,190 [Application Number 09/890,363] was granted by the patent office on 2006-08-29 for formulations comprising antisense nucleotides to connexins.
This patent grant is currently assigned to CoDa Therapeutics Ltd.. Invention is credited to David Laurence Becker, Colin Richard Green.
United States Patent |
7,098,190 |
Becker , et al. |
August 29, 2006 |
**Please see images for:
( Certificate of Correction ) ** |
Formulations comprising antisense nucleotides to connexins
Abstract
A therapeutic and/or cosmetic formulation comprising at least
one anti-sense polynucleotide to a connexin protein together with a
pharmaceutically acceptable carrier or vehicle is useful in site
specific down regulation of connexin protein expression,
particularly in reduction of neuronal cells death, wound healing,
reduction of inflammation, decrease of scar formation and skin
rejuvenation and thickening.
Inventors: |
Becker; David Laurence
(Langley, GB), Green; Colin Richard (Epsom,
NZ) |
Assignee: |
CoDa Therapeutics Ltd.
(Auckland, NZ)
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Family
ID: |
26652011 |
Appl.
No.: |
09/890,363 |
Filed: |
January 27, 2000 |
PCT
Filed: |
January 27, 2000 |
PCT No.: |
PCT/GB00/00238 |
371(c)(1),(2),(4) Date: |
November 02, 2001 |
PCT
Pub. No.: |
WO00/44409 |
PCT
Pub. Date: |
August 03, 2000 |
Foreign Application Priority Data
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Jan 27, 1999 [NZ] |
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333928 |
Oct 7, 1999 [NZ] |
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500190 |
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Current U.S.
Class: |
514/44A; 435/375;
536/23.1; 536/24.5; 536/24.1; 435/377 |
Current CPC
Class: |
A61P
17/00 (20180101); A61P 25/00 (20180101); C12N
15/111 (20130101); A61P 43/00 (20180101); C07H
21/04 (20130101); A61P 17/02 (20180101); A61K
31/711 (20130101); A61P 17/16 (20180101); C12N
15/1138 (20130101); A61P 29/00 (20180101); A61P
17/12 (20180101); A61K 38/00 (20130101); C12N
2310/11 (20130101); C12N 2320/32 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); A61K 31/70 (20060101); C07H
21/04 (20060101) |
Field of
Search: |
;435/375,377
;536/24.1,24.5,23.1 ;514/44 |
Foreign Patent Documents
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WO US96/19194 |
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Jun 1998 |
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WO |
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WO 9824797 |
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Nov 1998 |
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WO |
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Other References
Agrawal S., TIBTECH vol. 14: 376-387, Oct. 1996. cited by examiner
.
Branch A., TIBS vol. 23:45-50, Feb. 1998. cited by examiner .
Jen et al., Stem Cells vol. 18:307-319, 2000. cited by examiner
.
Qiu et al., Current Biology vol. 13: 1697-1703, Sep. 30, 2004.
cited by examiner .
Hodgins M., Journal of Investigative Dermatologyvol. 122(5):ix-x,
2004. cited by examiner .
R. Ruch et al., Molecular Carcinogenesis, 14:269-274 (1995). cited
by other .
J. Goliger et al., Molecular Biology of the Cell, 6:1491-1501
(1995). cited by other .
Moore et al., Am J. Physiology, vol. 265, No. 1, pp. C1371-C1388
(1994). cited by other .
Grazul-Bilska et al., Abstract, Biology Reproduction, vol. 58, No.
1, p. 78 (1998). cited by other .
European Patent Office, Search Report for Application No.
05016736.0, dated Dec. 19, 2005. cited by other .
Davis et al., "Modulation of Connexin43 Expression: Effects on
Cellular Coupling", 1995, U.S. cited by other .
Rozenthal et al., "Stable Transfection with Connexin43 Inhibits
Neuronal Differentiation of PC12 Cells", Oct. 25, 1997, U.S. cited
by other .
Becker et al., "Connexin Alpha1 and Cell Proliferation in the
Developing Chick Retina", Apr. 1999, U.S. cited by other .
Green et al., "Spatiotemporal Depletion of Connexins Using
Antisense Oligonucleotides", 2001, U.S. cited by other .
Frantseva et al., "Ischemia-Induced Brain Damage Depends on
Specific Gap-Junctional Coupling", Apr. 2002, U.S. cited by
other.
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Primary Examiner: McGarry; Sean
Attorney, Agent or Firm: Duane Morris LLP
Claims
The invention claimed is:
1. A method of treating a human subject having a wound, which
comprises administering to the wound a connexin 43 anti-sense
polynucleotide, whereby connexin 43 protein expression is
downregulated.
2. A method of reducing cell death resulting from a neuronal insult
to a human subject, which comprises administering to the site of
the neuronal insult a connexin 43 anti-sense polynucleotide,
whereby connexin 43 expression is downregulated.
3. A method according to claim 2 wherein the neuronal insult is to
the brain, spinal cord or optic nerve.
4. A method according to claim 2 in which said anti-sense
polynucleotide is administered in a sufficient amount to
downregulate connexin 43 expression for at least 24 hours
post-administration.
5. A method of promoting wound healing in a human which comprises
the step of administering to the wound an amount of a connexin 43
anti-sense polynucleotide effective to downregulate connexin 43
expression.
6. A method according to claim 1 or 5 in which the wound is the
result of trauma.
7. A method according to claim 6 in which trauma is a burn.
8. A method according to claim 1 or 5 in which the wound is the
result of a surgery.
9. A method of treating a human subject to reduce inflammation
associated with a wound or associated with a tissue subjected to a
physical trauma which comprises the step of administering to the
wound or tissue an amount of a connexin 43 anti-sense
polynucleotide effective to downregulate a connexin 43
expression.
10. A method according to claim 9 in which the tissue subjected to
physical trauma is selected from the group consisting of brain,
spinal cord and optic nerve.
11. A method of decreasing scar formation following a wound to a
human subject which comprises administering to the wound an amount
of a connexin 43 anti-sense polynucleotide effective to
downregulate a connexin 43 expression.
12. A method according to claim 1, wherein said anti-sense
polynucleotide is an oligodeoxynucleotide.
13. A method according to any of claims 1, 2, 5, 9, or 11 wherein
said connexin protein comprises the amino acid sequence coded for
by SEQ ID NO. 12.
14. A method according to any of claims 1, 2, 5, 9, or 11 wherein
said anti-sense polynucleotide is present in a composition
comprising a pharmaceutically acceptable carrier or vehicle.
15. A method according to claim 14, wherein said composition is
suitable for topical administration.
16. A method according to claim 14, wherein said composition is
formulated to provide sustained release of the antisense
polynucleotide.
17. A method according to claim 14, wherein said composition is
formulated to provide sustained release of the antisense
polynucleotide over at least 24 hours.
18. A method according to claim 13, wherein the anti-sense
polynucleotide is present in a composition comprising a
pharmaceutically acceptable carrier or vehicle formulated for
topical administration.
19. A method according to claim 13, wherein the anti-sense
polynucleotide is in the form of an impregnated dressing.
20. A method according to claim 14, wherein the pharmaceutically
acceptable carrier or vehicle is, or includes, a gel.
21. A method according to claim 20 in which the gel is a nonionic
polyoxyethylene-polyoxypropylene copolymer gel.
22. A method according to claim 14, wherein the composition further
includes a surfactant.
23. A method of decreasing cell death in a tissue of a mammal
comprising contacting the cells with an effective amount of a
connexin 43 antisense polynucleotide.
24. The method of claim 23, wherein said connexin 43 antisense
polynucleotide is an oligodeoxynucleotide.
25. The method of claim 24, wherein said oligodeoxynucleotide is an
unmodified phosphodiester oligomer.
26. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide binds to at least a portion of
a connexin 43 mRNA.
27. The method of claim 26, wherein said connexin 43 antisense
polynucleotide is exactly complementary to at least a portion of
said connexin 43 mRNA.
28. The method of claim 26, wherein said connexin 43 antisense
polynucleotide is not exactly complementary to at least a portion
of a connexin 43 mRNA.
29. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide is about 12 to about 40
nucleotides in length.
30. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide is about 30 nucleotides in
length.
31. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide comprises SEQ ID NO: 1.
32. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide comprises SEQ ID NO: 2.
33. The method of any of claims 1, 2, 5, 9, 11 or 23, wherein said
connexin 43 antisense polynucleotide comprises SEQ ID NO: 3.
34. The method of claim 23, wherein said connexin 43 is a human
connexin 43.
35. The method of claim 23, wherein said mammal is a human.
36. The method of claim 23, wherein said tissue is skin.
37. The method of claim 9 or 23, wherein said tissue is neural
tissue.
38. The method of claim 9 or 23, wherein said tissue is brain.
39. The method of claim 9 or 23, wherein said tissue is spinal
cord.
40. The method of claim 9 or 23, wherein said tissue is connective
tissue.
41. The method of any of claims 23 25, 34, 35 or 36, wherein said
connexin 43 antisense polynucleotide is administered to a
wound.
42. The method of claim 41, wherein said wound is a surgical
wound.
43. The method of claim 41, wherein said wound is a burn.
44. The method of any of claims 23 25, 34, 35 or 36, wherein said
connexin 43 antisense polynucleotide is administered to a site of
inflammation.
45. The method of any of claims 23 25, 34, 35 or 36, wherein said
connexin 43 antisense polynucleotide is disposed in a topical
formulation.
46. The method of claim 45, wherein said topical formulation
comprises a gel.
47. The method of claim 46, wherein said gel is a pluronic gel.
48. The method of any of claims 23 25, 34, 35 or 36, wherein said
connexin 43 antisense polynucleotide is administered by
syringe.
49. The method of any of claims 23 25, 27, 28 or 34 36, wherein
said connexin 43 antisense polynucleotide is administered as a
gel.
50. The method of any of claim 26, wherein said connexin 43
antisense polynucleotide is administered as a gel.
51. The method of any of claim 29, wherein said connexin 43
antisense polynucleotide is administered as a gel.
52. The method of any of claim 30, wherein said connexin 43
antisense polynucleotide is administered as a gel.
53. The method of any of claim 31, wherein said connexin 43
antisense polynucleotide is administered as a gel.
54. The method of any of claim 32, wherein said connexin 43
antisense polynucleotide is administered as a gel.
55. The method of any of claim 33, wherein said connexin 43
antisense polynucleotide is administered as a gel.
56. The method of any of claim 37, wherein said connexin 43
antisense polynucleotide is administered as a gel.
57. The method of any of claim 38, wherein said connexin 43
antisense polynucleotide is administered as a gel.
58. The method of any of claim 39, wherein said connexin 43
antisense polynucleotide is administered as a gel.
59. The method of any of claim 40, wherein said connexin 43
antisense polynucleotide is administered as a gel.
60. The method of any of claims 23 25, 27, 28 or 34 36, wherein
said connexin 43 antisense polynucleotide is administered as a
dressing.
61. The method of claim 26, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
62. The method of claim 29, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
63. The method of claim 30, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
64. The method of claim 31, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
65. The method of claim 32, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
66. The method of claim 33, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
67. The method of claim 37, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
68. The method of claim 38, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
69. The method of claim 39, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
70. The method of claim 40, wherein said connexin 43 antisense
polynucleotide is administered as a dressing.
Description
This application is a U.S. national stage application of
International Application No. PCT/GB00/00238, filed Jan. 27, 2000
(published as WO00/44409 on Aug. 3, 2000) and claims the benefit of
priority to NZ 333928 (filed Jan. 27, 1999) and NZ 500190 (filed
Oct. 7, 1999). The contents of each of which are hereby
incorporated in their entireties.
This invention relates to formulations for use in therapeutic
and/or cosmetic treatments, particularly those in which a localised
disruption in direct cell--cell communication is desirable.
BACKGROUND
Gap junctions are cell membrane structures which facilitate direct
cell--cell communication. A gap junction channel is formed of two
hemichannels (connexons), each composed of six connexin subunits.
These connexins are a family of proteins, commonly named according
to their molecular weight or classified on a phylogenetic basis ie.
into an .alpha. class and a .beta. class.
An ability to control connexin expression (and in particular to
down-regulate it) would therefore provide an opportunity to
modulate cell--cell communication within a patient for therapeutic
and/or remedial purposes. However, as a number of connexin proteins
are expressed widely throughout the body, a general downregulatory
effect is undesirable in inducing a therapeutic effect at a
specific site.
Anti-sense oligodeoxynucleotides (ODN's) have considerable
potential as agents for the manipulation of specific gene
expression (reviewed: Stein et al., 1992; Wagner 1994). However,
there remain difficulties which need to be overcome. These include
the short half life of such ODN's (unmodified phosphodiester
oligomers typically have an intracellular half life of only 20
minutes owing to intracellular nuclease degradation (Wagner 1994))
and their delivery consistently and reliably to target tissues.
It was with the intent of at least partially overcoming these
difficulties that the applicants devised the present invention.
SUMMARY OF THE INVENTION
Accordingly, in a first aspect, the invention provides a
formulation for use in therapeutic and/or cosmetic treatment, which
formulation comprises:
at least one anti-sense polynucleotide to a connexin protein;
together with a pharmaceutically acceptable carrier or vehicle.
In one preferred form, the formulation contains polynucleotides to
one connexin protein only. Most preferably, this connexin protein
is connexin 43.
Many aspects of the invention are described with reference to
oligodeoxynucleotides. However it is understood that other suitable
polynucleotides (such as RNA polynucleotides) may be used in these
aspects.
Alternatively, the formulation contains oligodeoxynucleotides to
more than one connexin protein. Preferably, one of the connexin
proteins to which oligodeoxynucleotides are directed is connexin
43. Other connexin proteins to which oligodeoxynucleotides are
directed include connexin 26, connexin 31.1 and connexin 32.
Conveniently, the oligodeoxynucleotide to connexin 43 is selected
from:
TABLE-US-00001 GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ ID
NO:1); GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ ID NO:2); and
GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT (SEQ ID NO:3)
Most conveniently, the oligodeoxynucleotide to connexin 43 is:
TABLE-US-00002 GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ ID
NO:1).
Conveniently, the oligodeoxynucleotide to connexin 26 is:
TABLE-US-00003 TCC TGA GCA ATA CCT AAC GAA CAA ATA (SEQ ID
NO:4).
Conveniently, the oligodeoxynucleotide to connexin 31.1 is:
TABLE-US-00004 CGT CCG AGC CCA GAA AGA TGA GGT C (SEQ ID NO:5).
Conveniently, the oligodeoxynucleotide to connexin 32 is:
TABLE-US-00005 TTT CTT TTC TAT GTG CTG TTG GTG A (SEQ ID NO:6).
The anti-sense polynucleotides may be formulated for parenteral,
intramuscular, intracerebral, intravenous, subcutaneous or
transdermal administration. The antisense polynucleotides are
preferably administered topically (at the site to be treated).
Suitably the antisense polynucleotides are combined with a
pharmaceutically acceptable carrier, vehicle or diluent to provide
a pharmaceutical composition.
Suitable pharmaceutically acceptable carriers or vehicles include
any of those commonly used for topical administration. The topical
formulation may be in the form of a cream, ointment, gel, emulsion,
lotion or paint. The formulation of the invention may also be
presented in the form of an impregnated dressing.
Suitable carrier materials include any carrier or vehicle commonly
used as a base for creams, lotions, gels, emulsions, lotions or
paints for topical administration. Examples include emulsifying
agents, inert carriers including hydrocarbon bases, emulsifying
bases, non-toxic solvents or water-soluble bases. Particularly
suitable examples include lanolin, hard paraffin, liquid paraffin,
soft yellow paraffin or soft white paraffin, white beeswax, yellow
beeswax, cetostearyl alcohol, cetyl alcohol, dimethicones,
emulsifying waxes, isopropyl myristate, microcrystalline wax, oleyl
alcohol and stearyl alcohol.
Preferably, the pharmaceutically acceptable carrier or vehicle is a
gel, suitably a nonionic polyoxyethylene-polyoxypropylene copolymer
gel, for example, a Pluronic gel, preferably Pluronic F-127 (BASF
Corp.). This gel is particularly preferred as it is a liquid at low
temperatures but rapidly sets at physiological temperatures, which
confines the release of the ODN component to the site of
application or immediately adjacent that site.
An auxiliary agent such as casein, gelatin, albumin, glue, sodium
alginate, carboxymethylcellulose, methylcellulose,
hydroxyethylcellulose or polyvinyl alcohol may also be included in
the formulation of the invention.
The pharmaceutical composition may be formulated to provide
sustained release of the antisense polynucleotide.
Conveniently, the formulation further includes a surfactant to
assist with oligodeoxynucleotide cell penetration or the
formulation may contain any suitable loading agent. Any suitable
non-toxic surfactant may be included, such as DMSO. Alternatively a
transdermal penetration agent such as urea may be included.
In a further aspect, the invention provides a method of
site-specific downregulation of connexin protein expression for a
therapeutic and/or cosmetic purpose which comprises administering a
formulation as defined above to a site on or within a patient at
which said downregulation is required.
In still a further aspect, the invention provides a method of
reducing neuronal cell death which would otherwise result from a
neuronal insult to a specific site in the brain, spinal cord or
optic nerve of a patient which comprises the step of administering
a formulation as defined above to said site to downregulate
expression of connexin protein(s) at and immediately adjacent said
site.
Preferably, the formulation is administered to reduce neuronal loss
due to physical trauma to the brain, spinal cord or optic
nerve.
Conveniently, the formulation is administered in a sufficient
amount to downregulate expression of said connexin protein(s) for
at least 24 hours post-administration.
In yet a further aspect, the invention provides a method of
promoting wound healing in a patient which comprises the step of
administering a formulation as defined above to said wound to
downregulate expression of connexin protein(s) at and immediately
adjacent the site of said wound.
Usually, the wound will be the result of trauma, including burns.
It may however be the result of surgery.
In yet a further aspect, the invention provides a method of
reducing inflammation as part of treating a wound and/or tissue
subjected to physical trauma which comprises the step of
administering a formulation as defined above to or proximate to
said wound or tissue.
Preferably, said wound is a burn.
Alternatively, said wound is the result of physical trauma to
tissue, including neuronal tissue such as the brain, spinal cord or
optic nerve.
In yet a further aspect, the invention provides a method of
decreasing scar formation in a patient who has suffered a wound
which comprises the step of administering a formulation as defined
above to said wound to down-regulate expression of connexin
protein(s) at and immediately adjacent the site of said wound.
Again, the wound may be the result of trauma or surgery, with the
formulation being applied to the wound immediately prior to
surgical repair and/or closure thereof.
In yet a further aspect, the invention provides a method of skin
rejuvenation or thickening for a cosmetic or therapeutic purpose
which comprises the step of administering, once or repeatedly, a
formulation as defined above to the skin surface.
Conveniently, said formulation includes oligodeoxynucleotides
directed to connexin 26 or connexin 43 and is administered to
regulate epithelial basal cell division and growth.
In another embodiment, said formulation includes
oligodeoxynucleotides directed to connexin 31.1 and is administered
to regulate outer layer keratinisation.
Preferably, the formulation is a cream or gel.
DESCRIPTION OF THE DRAWINGS
FIGS. 1 to 5 show sections of rat brain lesions treated with
Pluronic gel containing antisense oligodeoxynucleotides specific to
connexin 43, or for control lesions, Pluronic gel alone. In all
cases lesions were sectioned serially in a coronal plane and the
mid point sections used for analysis. Each image (except FIG. 5)
shows 4 mm by 5.33 mm of tissue. FIG. 5 is approximately 1.2 mm by
2 mm.
FIG. 1: FIGS. 1A and 1C show two side of a control lesion 24 hours
after lesioning. The lesion has been treated with Pluronic gel
alone. The sections have been Nissl stained (blue nuclei) and
antibody labelled with the neuronal marker Neuronal-N (brown
cells). FIGS. 1B and 1D show grey scale images of 1A and 1C
respectively with the outline of the lesion marked. Note the large
size of the lesion and the irregular spreading edges. The lesion
has spread downwards toward the corpus callosum (dashed line)
within just 24 hours of lesioning.
FIG. 2: A control lesion 24 hours after wounding. FIG. 2A shows
Nissl staining (blue nuclei) and Neuronal-N labelling of viable
neurons. FIG. 2B is a grey scale equivalent with the lesion edge
marked and the top of the corpus callosum marked (dashed line). The
original needle tract is clear but neuronal death has occurred well
back from the lesion edge as indicated by the Neuronal-N labelling.
The edges of the lesion are irregular and the lesion, within just
24 hours, has spread right down into the corpus callosum.
FIG. 3: FIGS. 3A and 3B are colour and grey scale images of a
connexin 43 antisense treated lesion, 48 hours after lesioning. The
lesion outline has been marked on FIG. 3B to show the extent of the
lesion and the top of the corpus callosum marked (dashed line).
FIG. 3A has been stained for Nissl (blue nuclei) and Neuronal-N
(pink cells). Note how compact the lesion is, even after 48 hours,
compared with control lesions (FIGS. 1 and 2). While there is some
spread to the right hand side, the left side of the lesion
essentially follows the original needle tract with little sign of
spreading. The left side of the lesion is very straight and it has
not spread down to the corpus callosum.
FIG. 4: FIGS. 4A and 4B show another connexin 43 antisense treated
lesion 48 hours after wounding. The labelling is the same as in
FIG. 3 with the lesion outlined on the grey scale image (FIG. 4B).
Even after 48 hours this lesion is extremely compact with slight
spreading only to the left (medial side). Note how straight the
right hand side of the lesion is with viable neurons right up to
the edge of the needle tract (and indeed surviving within the
lesioned area). The lesion is well above the corpus callosum
(dashed line) indicating virtually no downward spread.
FIG. 5: A higher magnification view showing the edge of a connexin
43 antisense treated lesion. The edge of the lesion has been marked
showing viable neurons (Neuronal-N labelled) right up to the edge
of the wounding needle tract even 48 hours after lesioning.
FIG. 6: GFAP (red) and connexin 43 (green) immunohistochemical
labelling of a connexin 43 specific antisense treated lesion, 24
hours after lesioning. The image is taken at the lateral edge of
the lesion at a point half way down the depth of the lesion.
Activated astrocyte levels are elevated compared with controls
(FIG. 7) and connexin 43 levels are markedly reduced. The connexin
labelling remaining is generally associated with blood vessels
(arrows).
FIG. 7: GFAP (red) and connexin 43 (green) immunohistochemical
labelling of a control lesion, 24 hours after lesioning. The image
is from the medial edge of the lesion and shows GFAP levels
slightly elevated over unlesioned cortex. Note the extensive
connexin 43 labelling, often co-localised with the GFAP astrocytic
marker (arrows).
FIG. 8 shows a comparison of lesion cross sectional lower half
areas 24 hours (circles) and 48 hours (diamonds) after lesioning.
The analysis was carried out on a mid section of serially sectioned
lesion cut on the coronal plane. Lesions were assessed using
Neuronal-N antibody labelling to delineate viable neurons. DB1
treated lesions (green markers) have been treated with antisense
oligodeoxynucleotides specific to connexin 43. The gel only lesion
group (red markers) also includes empty lesions while the HB3 group
(purple markers) are treated with gel containing random sequence
control oligodeoxynucleotides. Note that while connexin 43
antisense treated lesions can be large (presumably where the
antisense has not been well delivered), the smallest lesions are
all connexin 43 antisense treated. Lesions were made to a depth of
2 mm and analysis covers 1 mm and below so as to exclude the outer
edge where the antisense did not sit.
FIG. 9: Lesions in rat spinal cord 24 hours after treatment with
connexin 43 sense and antisense ODN's. The sense lesions were no
different from untreated controls whereas the antisense treated
lesions were smaller and with reduced inflammation.
FIG. 10: Lesions in neonatal mouse fore paws 24 hours after
treatment with connexin 43 sense ODNs (left paw) or antisense ODNs
(right paw). Note the reduction in inflammation and increased rate
of healing on the antisense treated paw.
FIG. 11: Sections through the centre of the 24 hour wounds shown in
FIG. 10. The sections have been stained with toluidine blue to
reveal neutrophils. There are significantly less neutrophils in the
antisense treated wound which was also less inflamed.
FIG. 12: Pairs of rat paw lesions five days after lesioning that
have been treated with connexin 43 specific antisense ODNs or sense
control ODNs. Antisense treated lesions are healing quicker and
show less signs of scarring.
FIG. 13: Pairs of rat paw lesions made at the neonate stage, and
viewed here 8 days after lesioning. Lesions were treated with
connexin 43 specific antisense or control sense ODN. Hair has grown
and it is clear that antisense treatment has resulted in smaller
scars and less hair loss. The site of the lesion remains prominent
in the sense treated control but is difficult to detect in the
antisense treated limb.
DESCRIPTION OF THE INVENTION
As broadly defined above, the focus of the invention is on
site-specific downregulation of connexin expression. This will have
the effect of reducing direct cell--cell communication at the site
at which connexin expression is down-regulated, which gives rise to
numerous therapeutic/cosmetic applications as described below.
The downregulation of connexin expression is based generally upon
the anti-sense approach using antisense polynucleotides (such as
DNA or RNA polynucleotides), and more particularly upon the use of
antisense oligodeoxynucleotides (ODN). These polynucleotides (eg.
ODN) target the connexin protein(s) to be downregulated. Typically
the polynucleotides are single stranded but may be double
stranded.
The antisense polynucleotide may inhibit transcription and/or
translation of the connexin. Preferably the polynucleotide is a
specific inhibitor of transcription and/or translation from the
connexin gene, and does not inhibit transcription and/or
translation from other genes. The product may bind to the connexin
gene or mRNA either (i) 5' to the coding sequence, and/or (ii) to
the coding sequence, and/or (iii) 3' to the coding sequence.
Generally the antisense polynucleotide will cause the expression of
connexin mRNA and/or protein in a cell to be reduced.
The antisense polynucleotide is generally antisense to the connexin
mRNA. Such a polynucleotide may be capable of hybridising to the
connexin mRNA and may thus inhibit the expression of connexin by
interfering with one or more aspects of connexin mRNA metabolism
including transcription, mRNA processing, mRNA transport from the
nucleus, translation or mRNA degradation. The antisense
polynucleotide typically hybridises to the connexin mRNA to form a
duplex which can cause direct inhibition of translation and/or
destabilisation of the mRNA. Such a duplex may be susceptible to
degradation by nucleases.
The antisense polynucleotide may hybridize to all or part of the
connexin mRNA. Typically the antisense polynucleotide hybridizes to
the ribosome binding region or the coding region of the connexin
mRNA. The polynucleotide may be complementary to all of or a region
of the connexin mRNA. For example, the polynucleotide may be the
exact complement of all or a part of connexin mRNA. However,
absolute complementarity is not required and polynucleotides which
have sufficient complementarity to form a duplex having a melting
temperature of greater than 20.degree. C., 30.degree. C. or
40.degree. C. under physiological conditions are particularly
suitable for use in the present invention.
Thus the polynucleotide is typically a homologue of the mRNA. The
polynucleotide may be a polynucleotide which hybridises to the
connexin mRNA under conditions of medium to high stringency such as
0.03M sodium chloride and 0.03M sodium citrate at from about 50 to
about 60 degrees centigrade.
The polynucleotide will typically be from 6 to 40 nucleotides in
length. Preferably it will be from 12 to 20 nucleotides in length.
The polynucleotides may be at least 40, for example at least 60 or
at least 80, nucleotides in length and up to 100, 200, 300, 400,
500, 1000, 2000 or 3000 or more nucleotides in length.
The connexin protein or proteins targeted by the ODN will be
dependent upon the site at which downregulation is to be effected.
This reflects the nonuniform make-up of gap junction (s) at
different sites throughout the body in terms of connexin sub-unit
composition. The connexin may be any connexin that naturally occurs
in a human or animal. The connexin gene (including coding sequence)
generally has homologue with any of the specific connexins
mentioned herein, such as homology with the connexin 43 coding
sequence shown in Table 2. The connexin is typically an a or
connexin. Preferably the connexin is expressed in the skin or
nervous tissue (including brain cells).
Some connexin proteins are however more ubiquitous than others in
terms of distribution in tissue. One of the most widespread is
connexin 43. ODN's targeted to connexin 43 are therefore
particularly suitable for use in the present invention.
It is also contemplated that ODN's targeted at separate connexin
proteins be used in combination (for example 1, 2, 3, 4 or more
different connexins may be targeted). For example, ODN's targeted
to connexin 43, and one or more other members of the connexin
family (such as connexin 26, 31.1, 32, 36, 40 and 45) can be used
in combination.
Individual antisense polynucleotides may be specific to a
particular connexin, or may target 1, 2, 3 or more different
connexins. Specific polynucleotides will generally target sequences
in the connexin gene or mRNA which are not conserved between
connexins, whereas non-specific polynucleotides will target
conserved sequences.
The ODN's for use in the invention will generally be unmodified
phosphodiester oligomers. They will vary in length but with a 30
mer ODN being particularly suitable.
The antisense polynucleotides may be chemically modified. This may
enhance their resistance to nucleases and may enhance their ability
to enter cells. For example, phosphorothioate oligonucleotides may
be used. Other deoxynucleotide analogs include methylphosphonates,
phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and
oligoribonucleotide phosphorothioates and their 2'-O-alkyl analogs
and 2'-O-methylribonucleotide methylphosphonates.
Alternatively mixed backbone oligonucleotides (MBOs) may be used.
MBOs contain segments of phosphothioate oligodeoxynucleotides and
appropriately placed segments of modified oligodeoxy- or
oligoribonucleotides. MBOs have segments of phosphorothioate
linkages and other segments of other modified oligonucleotides,
such as methylphosphonate, which is non-ionic, and very resistant
to nucleases or 2'-O-alkyloligoribonucleotides.
The precise sequence of the antisense polynucleotide used in the
invention will depend upon the target connexin protein. For
connexin 43, the applicant's have found ODN's having the following
sequences to be particularly suitable:
TABLE-US-00006 GTA ATT GCG GCA AGA AGA ATT GTT TCT GTC (SEQ ID
NO:1); GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ ID NO:2); and
GGC AAG AGA CAC CAA AGA CAC TAC CAG CAT (SEQ ID NO:3)
ODN's directed to other connexin proteins can be selected in terms
of their nucleotide sequence by any convenient, and conventional,
approach. For example, the computer programmes MacVector and
OligoTech (from Oligos etc. Eugene, Oreg., USA) can be used. For
example, ODN's for connexins 26, 31.1 and 32 have the following
sequences:
TABLE-US-00007 5' TCC TGA GCA ATA CCT AAC GAA CAA ATA (connexin 26)
(SEQ ID NO:4) 5' CGT CCG AGC CCA GAA AGA TGA GGT C (connexin 31.1)
(SEQ ID NO:5) 5' TTT CTT TTC TAT GTG CTG TTG GTG A (connexin 32)
(SEQ ID NO:6)
Once selected, the ODN's can be synthesised using a DNA
synthesiser.
For use in the invention, the ODN(s) require site-specific
delivery. They also require delivery over an extended period of
time. While clearly the delivery period will be dependent upon both
the site at which the downregulation is to be induced and the
therapeutic effect which is desired, continuous delivery for 24
hours or longer will often be required.
In accordance with the present invention, this is achieved by
inclusion of the ODN(s) in a formulation together with a
pharmaceutically acceptable carrier or vehicle, particularly in the
form of a formulation for topical administration.
Once prepared, the formulations of the invention have utility in
any therapeutic/cosmetic approach where a transient and
site-specific interruption in cell--cell communication is
desirable. These include in treating neuronal damage in the brain,
spinal cord or optic nerve (where the damage is to be localised as
much as possible), in the promotion of wound healing and in
reducing scar formation following, for example, cosmetic surgery or
burns.
In particular, topical formulations such as creams can be employed
to regulate epithelial basal cell division and growth (using ODN's
targeted to connexin 43) and outer layer keratinisation (using
ODN's targeted to connexin 31.1).
The antisense polynucleotides (including the ODN) may be present in
a substantially isolated form. It will be understood that the
product may be mixed with carriers or diluents which will not
interfere with the intended purpose of the product and still be
regarded as substantially isolated. A product of the invention may
also be in a substantially purified form, in which case it will
generally comprise 90%, e.g. at least 95%, 98% or 99% of the
polynucleotide or dry mass of the preparation.
Administration
The antisense polynucleotides (including ODN's) of the invention
(typically in the form of the formulation discussed herein) may
thus be administered to a human or animal in need of treatment,
such as a human or animal with any of the diseases or conditions
mentioned herein. The condition of the human or animal can thus be
improved. The polynucleotide and formulation may thus be used in
the treatment of the human or animal body by therapy. They may be
used in the manufacture of a medicament to treat any of the
conditions mentioned herein.
The antisense polynucleotides may be administered by typically (at
the site to be treated). Preferably the antisense polynucleotides
are combined with a pharmaceutically acceptable carrier or diluent
to produce a pharmaceutical composition. Suitable carriers and
diluents include isotonic saline solutions, for example
phosphate-buffered saline. The composition may be formulated for
parenteral, intramuscular, intracerebral, intravenous,
subcutaneous, or transdermal administration.
The dose at which an antisense polynucleotide is administered to a
patient will depend upon a variety of factors such as the age,
weight and general condition of the patient, the condition that is
being treated, and the particular antisense polynucleotide that is
being administered. A suitable dose may however be from 0.1 to 100
mg/kg body weight such as 1 to 40 mg/kg body weight.
Uptake of nucleic acids by mammalian cells is enhanced by several
known transfection techniques for example those including the use
of transfection agents. The formulation which is administered may
contain such agents. Example of these agents include cationic
agents (for example calcium phosphate and DEAE-dextran) and
lipofectants (for example lipofectam.TM. and transfectam.TM.).
The routes of administration and dosages described above are
intended only as a guide since a skilled physician will be able to
determine readily the optimum route of administration and dosage
for any particular patient and condition.
Homologues
Homology and homologues are discussed herein (eg. the
polynucleotides may be a homologue of sequence in connexin mRNA).
Such polynucleotides typically have at least 70% homology,
preferably at least 80, 90%, 95%, 97% or 99% homology with the
relevant sequence, for example over a region of at least 15, 20,
40, 100 more contiguous nucleotides (of the homologous
sequence).
Homology may be calculated based on any method in the art. For
example the UWGCG Package provides the BESTFIT program which can be
used to calculate homology (for example used on its default
settings) (Devereux et al (1984) Nucleic Acids Research 12, p387
395). The PILEUP and BLAST algorithms can be used to calculate
homology or line up sequences (typically on their default
settings), for example as described in Altschul S. F. (1993) J Mol
Evol 36:290 300; Altschul, S, F et al (1990) J Mol Biol 215:403
10.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information. This
algorithm involves first identifying high scoring sequence pair
(HSPs) by identifying short words of length W in the query sequence
that either match or satisfy some positive-valued threshold score T
when aligned with a word of the same length in a database sequence.
T is referred to as the neighbourhood word score threshold
(Altschul et al, supra). These initial neighbourhood word hits act
as seeds for initiating searches to find HSPs containing them. The
word hits are extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Extensions for the word hits in each direction are halted when: the
cumulative alignment score falls off by the quantity X from its
maximum achieved value; the cumulative score goes to zero or below,
due to the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLAST program uses as defaults a word length
(W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff
(1992) Proc. Natl. Acad. Sci. USA 89: 10915 10919) alignments (B)
of 50, expectation (E) of 10, M=5, N=4, and a comparison of both
strands.
The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 5873 5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
The homologous sequence typically differs from the relevant
sequence by at least (or by no more than) 2, 5, 10, 15, 20 more
mutations (which may be substitutions, deletions or insertions).
These mutations may be measured across any of the regions mentioned
above in relation to calculating homology.
The homologous sequence typically hybridises selectively to the
original sequence at a level significantly above background.
Selective hybridisation is typically achieved using conditions of
medium to high stringency (for example 0.03M sodium chloride and
0.03M sodium citrate at from about 50.degree. C. to about
60.degree. C.). However, such hybridisation may be carried out
under any suitable conditions known in the art (see Sambrook et al
(1989), Molecular Cloning: A Laboratory Manual). For example, if
high stringency is required, suitable conditions include
0.2.times.SSC at 60.degree. C. If lower stringency is required,
suitable conditions include 2.times.SSC at 60.degree. C.
Various aspects of the invention will now be described with
reference to the following experimental section which will be
understood to be provided by way of illustration only and not to
constitute a limitation on the scope of the invention.
Experimental
Experiment 1
Materials and Methods
Antisense Application
30% Pluronic F-127 gel (BASF Corp) in phosphate buffered saline
(molecular grade water) was used to deliver unmodified a1 connexin
(connexin 43) specific anti-sense ODN's to the developing chick
embryo (Simons, et al., 1992). Chick embryos were incubated at
38.degree. C. and staged according to Hamilton and Hamburger
stages. Eggs were windowed and the vitleline and amniotic membranes
over the area to be treated were opened using fine forceps. After
anti-sense application eggs were sealed with tape and replaced in
the incubator for 48 hours at which time most experiments were
analysed, the exception being for the time course analysis of a1
connexin "knockdown" and recovery.
Pluronic gel is liquid at low temperatures, 0 4.degree. C., but
sets when dropped onto the embryo at physiological temperature,
remaining in place for at least 12 hours. The gel has the
additional advantage of being a mild surfactant and this, used
either alone or in conjunction with DMSO, appeared to markedly
expedite ODN penetration into cells (Wagner, 1994). Addition of an
FITC tag to DB1 ODN, viewed using confocal laser scanning
microscopy, demonstrated intracellular penetration of the probes.
Sequences of deoxyoligonucleotides used are shown in Table 1.
Antisense oligodeoxynucleotides to Connexin 43
TABLE-US-00008 DBI GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (SEQ ID
NO:2) CGI GGC AAG AGA CAC CA AGA CAC TAC CAG CAT (SEQ ID NO:3)
Control oligodeoxynucleotides
TABLE-US-00009 DBI(sense) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC
(SEQ ID NO:7) DBI(chick) GTA GTT ACG ACA GGA GGA ATT GTT CTC GTC
(SEQ ID NO:8) CV3(random) TCG AAC TGT CAA GAC TGC TAT GGC GAT CAT
(SEQ ID NO:9)
Gel Only
All ODN's were applied at 0.5 1.0 .mu.M final concentration
following dose dependent analysis during preliminary experiments
covering a range of concentrations from 0.05 .mu.M to 50 .mu.M.
General toxicity effects only became apparent with ODN
concentrations greater than 10 .mu.M. ODN gel mixtures were
prepared from concentrated stock solutions stored at -80.degree.
C.
Anti-Sense Sequences
DB1 is a mouse anti-sense sequence, complementary to bases 1094
1123 of the a1 connexin gene. It has four mismatches with chick a1
connexin sequence. CG1 is complementary to chick a1 connexin bases
720 749. Efficacy of this probe was improved with 1%
Dimethylsulphoxide (DMSO) added to the gel. DMSO had no added
effect on other anti-sense ODN or control results.
Control Sequences
DB1 (Chick) is the chick a1 connexin equivalent of DB1 matching
chick a1 connexin bases 954 983. Analysis however, indicates a high
probability of forming stem loop structures (G=-7.0 kcal/mol. Loop
Tm=92.degree.) and homodimerisation (Tm=1.5.degree.) and therefore
acts as a control sequence. It has been reported that some sense
oligonucleotides can form stable DNA triplets (Neckers et al. 1993)
inhibiting transcription. However, this was not apparent with DB1
(sense). A random control sequence with no stable secondary
structure (G=1.4 kcal/mol) and unstable homodimerisation was also
used, called CV3. An additional control applying equal
concentration mixture of DB1 and DB1 (sense) gave background levels
of defects.
Monitoring of Protein Knockdown
Immunohistochemical localisation of a1 connexin gap junction
protein at cell--cell interfaces provides a direct measurement of
the anti-sense effect. Anti-peptide a1 connexin specific antibody
probes were used to stain wholemount embryos and the connexin
distribution was analysed using confocal laser scanning microscopy
according to established procedures (Green et al. 1995). Control
labelling for two other connexins expressed in the developing chick
embryo (connexins b1 & b2) was similarly carried out, also
using sequence specific antibodies (Becker et al., 1995).
Results
Reduction of a1 Connexin Expression
Using Pluronic F-127 gel to deliver unmodified a1 connexin specific
anti-sense ODN's to the developing chick embryo, protein expression
can be interfered with at chosen time points and allows the
anti-sense treatment to be targeted to specific regions of a chick
embryo. A droplet of gel containing the anti-sense at a relatively
low concentration was placed precisely onto individual embryos. The
gel sets and remains in place for at least 12 hours and thus a
sustained low dose of anti-sense is maintained in this region. The
anti-sense applications were targeted and timed to block junction
formation prior to the periods of elevated expression in the limb,
neural tube and face. These times were chosen to optimise the
effects of the anti-sense by reducing the expression of new protein
rather than being dependent upon the turnover of protein already in
the membranes of the cells of the target tissue. Both DB1 and CG1
ODN's reduced expression of a1 connexin protein within two hours in
the neural tube and limb bud, dramatic within 4 8 hours and
persisted at 18 24 hours and 48 hours in some tissues (data not
shown). No down regulation of a1 connexin protein was evident in
any of the controls used. Equally, two other members of the
connexin family expressed in the chick embryo, b1 connexin and b2
connexin, were unaffected by the a1 connexin specific anti-sense
ODN.
Several parallel controls were run with all of the experiments.
These included; DB1 sense, DB1 anti-sense and DB1 sense combined,
DB1 chick (which forms stem loop structures with itself), random
ODN's CV3, Pluronic gel alone, Pluronic gel with DMSO and PBS
alone). None of the controls had a noticeable effect on a1 connexin
protein expression.
Experiment 2
Introduction
Astrocytes constitute the most abundant cell type in the mammalian
brain. They are extensively coupled to one another and to neurons
through gap junctions composed predominantly of connexin 43 (Giaume
and McCarthy (1996)). Following ischaemia induced or physical brain
damage these channels remain open and a spreading wave of
depression (initiated by raised interstitial potassium and
glutamate and apoptotic signals) is propagated (Cotrina et al.,
(1998); Lin et al (1998)). Waves of increased cytosolic calcium and
second messenger molecules such as IP3 are slowly spread via the
gap junction channels to neurons beyond the core of the damaged
region, resulting in lesion spread in the 24 48 hours following the
insult. In this manner, undamaged neighbouring cells are destroyed
(Lin et al., 1998), the so-called bystander effect.
This experiment investigates the ability of the formulations of the
invention to prevent this bystander effect.
Materials
Oligodeoxynucleotides were prepared with the following
sequences:
TABLE-US-00010 GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (connexin
43) (SEQ ID NO:2) TTG TGA TTT ATT TAG TTC GTC TGA TTT C (random
control) (SEQ ID NO:10)
Methods Oligodeoxynucleotides (ODN's)
Unmodified ODN's were delivered in Pluronic F-127 gel (BASF,
Germany) in phosphate buffered saline (PBS). Pluronic gel is liquid
at low temperatures (0 4.degree. C.) and sets at physiological
temperatures, and is also a mild surfactant. Unmodified ODN's
normally have a half life of approximately 20 min in cells (Wagner,
1994) but the Pluronic gel loading method provides a continual
diffusion source, the gel acting as a reservoir Becker et al.,
(1999)). ODN's specific to connexin 43 were applied, or control
random ODN's of similar base composition, at 2 .mu.M final
concentration. Gel only controls were also carried out. ODN's were
30 mers analysed to show that no hairpin looping or
homodimerisation should occur.
Lesioning
Brain lesions were carried out on 250 300 g male Wistar rats.
Animals were anaesthetised with 1 2% halothane in oxygen and the
head held in a steriotaxic clamp. The region around the lesion site
was shaved and the skin over the skull slit in a sagtital plane
with a scalpel and pulled back to leave the skull plates clear. A
0.5 mm diameter hole was drilled through the skull plate 3 mm to
the right of bregma using an Arlec engraver and a lesion made into
the cortex of the brain using a 19G 11/2 gauge syringe needle
attached to a micrometer stage. The stage allowed accurate
directional control and a precise 2 mm penetration depth which kept
the lesion within the cortex and well above the corpus
callosum.
With the animal prepared. 10 ml of ice cold Pluronic F-127 gel
(BASF) containing connexin 43 specific ODN (or a control ODN) was
sucked into a precooled 19 G 11/2, gauge syringe needle filed off
so as to have a flat tip. The syringe needle was attached to a
volumetric pipette via a cut down yellow pipette tip. The gel then
set in the needle as it warmed to room temperature. The needle with
the gel plug at its tip was transferred to a 1 ml syringe
containing PBS and a sleeve slipped over the needle shaft so that
the needle tip could be lowered into the lesion with the sleeve
(coming up against the skull) preventing overpenetration. Gentle
pressure on the syringe plunger "popped" the gel plug out of the
needle into the lesion. The wound was then treated with hydrogen
peroxide to stop bleeding and the skin sutured back into place.
Animals were carefully monitored and left until ready for sacrifice
24 hours, 48 hours or 12 days later.
Frozen Sectioning
Animals were sacrificed using Nembutal (pentobarbitone sodium,
Virbac) and decapitated. The brains were removed intact and
immediately frozen in dry ice snow and stored at -80.degree. C.
until ready for sectioning. Serial cryosections (30 mm sections)
were taken from front to rear (coronal plane), the sections dry
mounted onto chrome alum treated slides, and stored for
histochemistry or immunohistochemistry at -80.degree. C. The first
and last section of each lesion was recorded so that the mid-point
sections of the lesion were clearly identified.
Histochemistry
For haemotoxylin and eosin staining sections were hydrated through
a descending series of alcohols (absolute, 2.times.95%, 1.times.70%
and water) and stained in Gill's haemotoxylin for 4 minutes. The
sections were then washed in water, dipped in Scott's water and
rewashed in water. They were then stained for 30 seconds in Moore's
buffered eosin. The sections were washed once more in water before
dehydration through a series of alcohols (2.times.95%, 1.times.
absolute), 50:50 alcohol:xylol and dipped in xylene. The sections
were then mounted using Histomounta mounting medium.
For Nissl staining, sections were dehydrated in an ascending graded
series of alochols (75%, 95%, 3.times.100%), five minutes in each,
and defatted in xylene for five minutes. The sections were then
rehydrated by descending through the same series of alcohols and
washed in water. The sections were then placed in a Nissl staining
solution (5 ml of a 2% aqueous Cresyl violet stock solution, 90 ml
of a 6% glacial acetic acid in water solution, 10 ml of a 1.35%
sodium acetate solution) for 10 minutes. The sections were then
quickly dehydrated in a series of ascending alcohols for 5 minutes
at 75%, then 2 minutes each at 95% and 3.times.100%, three charges
of xylene for 10 minutes each. They were then coverslipped with
Histomounta mounting medium.
Immunohistochemistry
Frozen sections were first allowed to come back up to room
temperature in PBS. They were then permeabilised in methanol for
two minutes, rinsed in PBS and transferred to a solution of 0.1M
lysine and 0.1% Triton-X 100 in PBS for blocking over 30 min. Two
washes in PBS, each of two minutes, followed. PBS was removed and
50 ml per section of primary antibody was applied.
Immunohistochemistry was carried out with primary antibodies
against connexin 43, Neuronal-Nuclei (vertebrate specific nuclear
protein NeuN) and GFAP (glial fabrillary acidic protein). The
following antibodies were used:
Rabbit anti-Cx 43 (Gourdie et al., (1991)) at a concentration of
1:300.
Mouse anti-Cx 43 (Chemicon International, Inc.) at a concentration
of 1:100.
Rabbit anti-rat GFAP (DAKO, Z0334), at a concentration of
1:1000.
Mouse anti-Neuronal Nuclei (Chemicon International, Inc.) at a
concentration 1:1000.
For connexin and GFAP labelling sections were incubated overnight
at 4.degree. C. They were then washed three times 15 minutes in PBS
on an orbital shaker. Following this, excess PBS was removed and 50
ml per section of Alexaa 488 anti-rabbit IgG (Molecular Probes,
Oregon, USA) was applied at a concentration of 1:200. For
monoclonals and double labelling a CY3 (Chemicon, 132C) anti-mouse
secondary antibody was used. Sections were incubated in the dark
for two hours at room temperature followed by three washes of 15
minutes in PBS. For mounting excess PBS was removed from the slides
and one or two drops of Citifluor (glycerol/PBS solution) anti-fade
medium was applied. A coverslip was lowered onto the sections and
sealed with nail varnish. For Neuronal-N labelling the secondary
antibody was a biotinylated Goat anti-mouse followed by an avidin
linked HRP and DAB reaction (Sigma ExtrAvidin or DAKO Quickstain
kit).
Imaging and Analysis
Immunofluorescent labelling was carried out using a Leica TCS 4D
confocal laser scanning microscope. Double labelled images were
subsequently combined using the Leica Combine function or in Adobe
Photoshop. Haemotoxylin and eosin, and Nissl stained samples or
Neuronal-N labelled sections were captured using a Kontron (Zeiss)
Progress 3008 digital camera and lesion areas analysed using
MetaMorph (Universal Imaging Corp). Lesion areas were analysed for
the middle section of each lesion.
Results
The well documented spread of brain lesions in the first 24 48
hours after trauma occurred in our control gel experiments and all
lesions, controls and antisense treated, tended to spread near the
outer edge where the gel is less likely to sit after loading.
However, control lesions spread downwards into the corpus callosum
and sideways to form ragged, spreading edges (FIGS. 1 and 2).
Examination of Neuronal-N antibody labelled tissues reveals
neuronal death occurring well back from the lesion edge, with areas
of Nissl staining in which no viable neurons remain. This spread
occurs predominantly within 24 hours (FIGS. 1 and 2), continuing up
to 48 hours after lesioning. This is especially apparent in FIG. 2
where neuronal death is evident within 24 hours well back from the
lesion edge into otherwise normal looking tissue, and the lesion
has spread right down into the corpus callosum. In contrast, the
better connexin 43 antisense treated lesions remain confined to the
original lesion site and have clearly defined base levels (FIGS. 3
and 4). Neuronal-N labelling colocalises with Nissl stained tissue
and none of the connexin 43 antisense treated lesions spread
through the corpus callosum. Neuronal-N labelling shows neuronal
survival right up to the edge of the original needle tract lesion.
Surviving neurons around these lesions often define sharp
boundaries marking the edge of the needle tract (FIGS. 3 and 5).
More tissue remains viable within the lesion itself after antisense
treatment; in control lesions cell death leads to tissue loss
within the lesion area (compare control lesion in FIG. 2 at 24
hours with antisense treated lesions in FIGS. 3 and 4 at 48
hours).
While antibody labelling of glial fibrillary acidic protein (GFAP)
shows some increased astrocyte activation at the edges of lesions,
connexin 43 protein levels are clearly reduced at many places along
the edge of antisense treated lesions, particularly the basal and
medial edges (FIG. 6) compared with controls (FIG. 7). In some
areas the only connexin 43 labelling remaining 24 hours after
connexin 43 specific antisense treatment is in blood vessel walls
despite raised GFAP levels (FIG. 6). In general, connexin 43
labelling around antisense treated lesion collocalises to a much
lesser extent with GFAP labelling than in controls in which over
half of the connexin 43 labelling is astrocyte related. Other
connexin levels (connexins 26 and 32) did not appear to be altered
by the connexin 43 specific antisense treatments.
36 animals were lesioned. Cross sectional area (central slice of
the lesion volume in a coronal plane) was analysed for 21 animals.
The results are shown in Table 2.
TABLE-US-00011 TABLE 2 Cross sectional areas of lesions treated
with control and connexin 43 specific oligodeoxynucleotides, left
empty, or treated with gel only. Measurements are for animals
measured after 24 hours, 48 hours and 12 days. Two sets of figures
are included - measurements of the entire lesion, and measurements
from 1 mm below the surface. In analysis of the second group the
largest DB1 treated lesion (brackets) is excluded as it falls
outside 3 standard deviations from the mean for this group. Note
that the rat brain does heal (unlike other species) and 12 days
lesion measurements do not represent the original extend of lesion
spread. DB1 is anti connexin 43 treated HB3 is random oligo and
appears to be toxic Entire Lesion: (measurements in square mm) 24
hours 48 hours 12 days DB1 2.42; 3.16; 3.78; 5.57 3.7; 6.05; 2.91;
3.41; 2.79; 2.86 4.53 HB3 7.14 13.19 Gel/empty 5.04; 4.48 3.96;
3.41; 3.56; 5.91 2.58; 3.3 Lesions from 1 mm down: (this is
considered a more accurate measure as all lesions tend to spread at
the outer lip indicating that the treatment gel has settled in the
bottom of the lesion and/or the outer cortex has been damaged when
drilling the skull or inserting the gel loading needle). 24 hours
48 hours 12 days DB1 0.91; 1.13; 2.12; 2.41 (3.38); 0.99; 1.54;
1.44; 0.47; 1.2 1.08 HB3 5.9 5.6 Gel/empty 3.2; 2.19 1.86; 1.5;
1.68; 2.17 1.07; 1.43
In the final analysis the lesion area from a line 1 mm below the
outer cortex edge was measured so as to exclude lesion spread at
the outer edge where antisense treatments have little or no effect
(owing to gel being injected into and settling at the bottom of
lesions). One antisense treated animal falls more than three
standard deviations outside the mean for this group and has been
excluded. Mean lesion size for antisense treated lesions at 24 and
48 hours was 1.45 mm.sup.2 (+/-0.55), for controls 2.1 mm.sup.2
(+/-0.6). The four smallest (of 8 antisense treated and 8 control
lesions at 24 and 48 hours) were all connexin 43 antisense treated
with the smallest control lesion 50% larger than these four. This
data is also shown in graphical form in FIG. 8. By 12 days
regeneration occurs in the rat (but not in human brain tissue) and
the limits of lesion spread are not clearly defined.
Discussion
The Pluronic gel plug--antisense ODN method has been used to study
the effect of connexin 43 knockdown during astrocytosis which
occurs following lesioning of the cerebral cortex of the mammalian
brain. In the brain, release of toxins from dying neurons causes
what is known as the bystander effect, with the toxins spreading to
neighbouring cells through gap junction channels (Lin et al,
(1998)). Under neurodegenerative conditions, slow release of toxins
apparently leads to an upregulation of connexin 43 channels in
astrocytes to enable the transport and removal of the toxins to the
blood stream. In cases of severe trauma however, this upregulation
aids the spread of high toxin levels to neighbouring neurons,
killing them. Blocking of the connexin 43 upregulation and
knockdown of connexin 43 channels prevents this spread leading to
lesions up to 50% smaller in cross sectional area. This has
significant implications in the management of ischeamic stroke,
treatment of neurodegenerative diseases, and modulation of side
effects from surgical intervention.
Experiment 3
Introduction
The bystander effect in neural tissues whereby damaged neurons
release toxins which spread and kill neighbouring cells is well
documented. Experiment 2 shows that this effect can be reduced in
the brain using an antisense oligodeoxynucleotide sustained release
approach to knockdown the gap junction protein connexin 43.
Another tissue of similar composition to the brain is the spinal
cord in which the neural population is supported by populations of
glial cells, including astrocytes which are responsible for the
neuroprotective effect by removing glutamate and excess calcium
from the neural environment. This experiment investigates the
ability of the formulations of the invention to reduce the spread
of spinal cord lesions.
Materials
Oligodeoxynucleotides were prepared with the following
sequences:
TABLE-US-00012 GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (connexin
43) (SEQ ID NO:2) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC (sense
control) (SEQ ID NO:7)
Methods
Wistar rats were anaesthetised and their spinal cord exposed. A
standard hemisection lesion was then made in the cord and 5 ml of
chilled Pluronic gel, containing either antisense or sense ODN's to
connexin 43 (5 .mu.M) was placed in the lesion. Applications were
made blind. The exposed cord was then recovered and the rat
returned to its cage. Some animals were sacrificed at 24 hours
whereas others were maintained for 12 days and two months in order
to determine the extent of neuronal regeneration and the final size
of the lesion. For axonal regeneration studies the rats were
anaesthetised and their axons severed prior to their entry site to
the spinal cord. A pellet of Horse radish peroxidase (HRP) was
placed in the cut in order to retrogradely label the axons over a
24 hour period. Next day the rats were sacrificed and their spinal
cords removed and fixed in 2% paraformaldehyde. Cords were then
processed for cryosectioning and serial longitudinal 8 mm sections
were taken through the cords. Sections were then immunostained for
either connexins or GFAP along with propidium iodide as a nuclear
marker, or processed to reveal the HRP.
Results
At 24 hours post lesion there was a marked difference between the
spinal cord lesions treated with connexin 43 sense and antisense
ODN's. The sense lesions appeared no different from untreated
controls whereas the antisense treated lesions appeared smaller and
less inflamed (FIG. 9).
At 12 days HRP labelled axons could be seen in both sense and
antisense treated cords but in neither case did significant numbers
of regenerating axons cross the lesion. However, there was a marked
difference in lesion size with the antisense lesion appearing
significantly smaller than the sense or untreated lesions.
Two months after lesioning the spinal cords HRP labelling of
regenerating axons revealed that they had failed to cross the
lesion site in both sense and antisense treatments. Lesion size was
significantly smaller in antisense treated cords indicating a
significant reduction in secondary neuronal cell death.
Discussion
Using the formulations of the invention, the antisense
oligodeoxynucleotide knockdown of connexin 43 significantly reduces
the lesion spread which occurs in the first 24 48 hours after
spinal cord injury. The knockdown of connexin 43 also reduces
inflammation, further aiding in the neuroprotective effect, but
there was no change in the ability for neurons to grow back across
the lesion site. Thus, antisense treatment with connexin 43
specific oligodeoxynucleotides cannot aid regrowth of damaged
neurons, but has a significant neuroprotective effect reducing the
spread of the insult.
Experiment 4
Introduction
To repair skin wounds a number of cell types, such as fibroblasts,
endothelial cells and keratinocytes are activated to proliferate,
migrate and lay down extracellular matrix to fill the wound.
Communication and intercellular signalling is a key feature of the
wound healing process. Extracellular signalling mechanisms are
thought to be the key players though it is also probable that
intercellular signalling through the extensive networks of gap
junction channels in the skin layers may also have a role. Calcium
waves spreading away from injured cells through the epidermis may
signal their damage. In normal wound healing connexin levels start
to fall within 6 hours and take up to 6 days to recover. The roles
that these changes play are not understood but one theory is that
cells are released from their neighbours to divide rapidly, and
then junctions reform to coordinate migration into and over the
wound site.
This experiment investigates the ability of the formulations of the
invention to effect wound healing.
Materials
Oligodeoxynucleotides were prepared with the following
sequences:
TABLE-US-00013 GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (connexin
43) (SEQ ID NO:2) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC (sense
control) (SEQ ID NO:7)
Methods
Neonatal mice, CD1 strain, were anaesthetised with local
anaesthetic by spray. A clean incision wound, 2 mm long, was then
made along the length of both fore paws with an iridectomy knife.
By making the wounds under a dissecting microscope they can be made
very reproducible in size. They generally heal in 3 6 days. Carbon
powder was dusted into the wounds in order to mark them for
subsequent identification of the wound site at late time
points--this does not affect the healing in any way. 5 ml of
chilled Pluronic gel, containing either Sense or Antisense ODN's
was then applied to the wounds. The Pluronic gel is liquid between
0 4.degree. C. but sets at higher temperature. Once applied to the
wound the gel sets in place and acts as a slow release reservoir
for the ODN's as well as a mild surfactant, aiding the penetration
of ODN's into the tissue. Application of Sense ODN's was made to
one paw and Antisense to the other, alternating left and right
between litters. Pups were warmed under a lamp and then returned to
their mother. Wounds were examined daily and scored for quality of
healing. Representative pups were selected at 1 day, 5 day and 8
day post operation and their forelimbs photographed before the pups
were anaesthetised and perfused with 2% paraformaldehyde. The
forelimbs were removed and immersion-fixed in 2% paraformaldehyde
overnight and then processed for resin (1 day) or wax (2 days
onward) histology.
Inflammation of the wound was assessed 24 hours after wounding.
Resin sections through the wound are stained with Toluidine blue to
reveal nissl positive cells, neutrophils, which are the first cells
to respond to injury. These can also be revealed using neutrophil
specific markers.
Cell death and clearance is assessed by Tunel labelling to
determine the rate of clearance of apoptotic cells. Macrophage
staining was used to show the period of clearing up following cell
death. These are carried out days 3 5 post wounding.
Angiogenesis
Granulation is a feature of healing connective tissue and is cased
by the invasion of numerous capillaries. Macrophages are known to
express potent angiogenic factors such as VEGF. The degree of
vascularisation is monitored with antibodies to VEGF receptors,
anti-PCAM and anti-flt-1 which are both good blood vessel markers.
Contraction of this tissue is brought about by the differentiation
of wound fibroblasts into a contractile myofibroblast. After they
have pulled the wound together they die apoptotically and are
removed by macrophages. These cells can be revealed by smooth
muscle actin specific antibodies and their formation and removal
followed.
Hyperinnervation
Sensor, nerves are very sensitive to the signals released on
wounding and show transient sprouting at the sites of adult wounds.
However, in neonatal wounds this sprouting is more profuse and
results in permanent hyperinnervation. Whilst it is not clear what
these signals are it is likely that they are released from
inflammatory macrophages. Hyperinnervation is maximal at 7d post
wounding and nerve distribution can be revealed using PGP 9.5
antibody against neurofilaments.
Scarring is normally assessed weeks or months after closure of the
wound. However, a reasonable assessment can be made 12 days after
wounding. Sections through the wounds are stained with the collagen
stain Picrosirus Red and examined on a confocal microscope to
determine the collagen density and orientation at the wound
site.
Results
1 Day
At 24 hours after wounding marked differences were apparent between
the sense and antisense treated limbs. Sense treated wounds looked
no different from untreated with a normal spectrum of healing
grades and rates (FIG. 10). Antisense treated limbs were markedly
different from the controls, they appeared to be less inflamed and
the healing rate was generally faster.
Resin sections of representative limbs stained with a nissl stain
revealed significantly less neutrophils cells indicating a less
inflamed tissue (FIG. 11).
5 Days
By days after wounding scabs had started to fall off. At this stage
most of the antisense treated wounds appeared to be smaller than
the sense treated With either small scabs or less prominent
scarring (FIG. 12).
8 Days
8 days after wounding the limbs had grown hair. Sense treated
wounds were still visible being demarcated by a lack of hair around
the wound site. Antisense treated wounds were mostly invisible
being covered by normal hair growth. This difference in hair growth
indicates reduced scarring has occurred in the antisense treated
wounds (FIG. 13).
Conclusions
Application of connexin 43 antisense ODN's to a wound has a marked
affect on the healing process. The first noticeable effect is a
reduction in the inflammation of the wounds which is noticeable in
sections which show a much lower inflammatory response in terms of
levels of neutrophils. As healing progresses, antisense treated
wounds heal faster and with less scarring than control lesions.
This reduction in inflammatory response and subsequent improved
healing is possibly owing to reduced neutrophil communication and
to a speeding up of natural healing processes. The antisense ODN's
can reduce connexin expression in 4 8 hours so they will not have
an effect on the initial signalling of wounding but play a role in
the secondary signalling events. It is interesting to note that
neutrophils which invade in response to the wounding normally
express large amounts of connexin 43. It is also possible that they
form gap junctions with other cells in the wound and communicate
with them. Reduction in this form of communication may result in a
reduction of secreted factors from the neutrophils and may reduce
cell death in the wound as well as granulation and
hyperinnervation. It is also known that under normal conditions
connexin protein levels (connexins 26, 31.1 and 43) are reduced in
both the epithelial and subdermal layers of wounds starting within
6 hours, and remaining lowered for up to 6 days. The antisense
approach may speed up this initial protein reduction by blocking
translational processes as protein removal from the membrane is
occurring. Certainly, the effects of connexin 43 knockdown
immediately following wounding has marked effects on reducing
inflammatory levels and increasing healing rates.
Experiment 5
Introduction
The inflammation and secondary cell death that follows burning is
of major concern. Victims of severe burns over a high percentage of
their body often die one or two days after trauma. This experiment
investigates the ability of the formulations of the invention to
beneficially affect the burn recovery process.
Materials
Oligodeoxynucleotides were prepared with the following
sequences:
TABLE-US-00014 GTA ATT GCG GCA GGA GGA ATT GTT TCT GTC (connexin
43) (SEQ ID NO:2) GAC AGA AAC AAT TCC TCC TGC CGC AAT TAC (sense
control) (SEQ ID NO:7)
Methods
Reproducible burns are delivered to moistened skin, and Pluronic
gel containing antisense ODN's injected subdermal to the burn. A
series of burns were made using a soldering iron to the left and
right sides of the skull of six newborn mice. The burns on one side
of the head were treated with connexin 43-specific ODN in Pluronic
gel and those on the other side with sense control ODN in Pluronic
gel.
Results
After 24 hours, all six connexin 43 ODN treated burns showed lower
levels of inflammation compared with the control burns. These
differences were marked (data not shown).
Utility
Thus, in accordance with the invention, there are provided
formulations by which cell--cell communication can be downregulated
in a transient and site-specific manner. The formulations therefore
have application in methods of therapy and in cosmetic
treatments.
The delivery of the ODN component of the formulation for an
extended period (24 hours or longer) is a particular advantage in
treating neuronal damage. This is because, in most instances of
direct physical neuronal insult, neuronal cell loss extends well
beyond the site of actual injury to the surrounding cells. This
secondary neuronal cell loss occurs within 24 hours of the original
injury and is mediated by junction gap cell--cell communication.
Downregulation of connexin protein expression therefore blocks or
at least downregulates communication between the cells and
minimises secondary neuronal cell damage.
Equally, in instances of other tissue damage (particularly wounds)
the formulations of the invention have been found effective in both
promoting the wound healing process, reducing inflammation and in
minimising scar formation. The formulations therefore have clear
benefit in the treatment of wounds, whether the result of external
trauma (including burns) or surgical intervention.
It will further be appreciated that the above description is
provided by way of example only and that modifications can be made,
both in terms of the specific ODN's and pharmaceutically acceptable
carriers or vehicles employed without departing from the scope of
the present invention.
TABLE-US-00015 TABLE 3 (SEQ ID NO:12) 1 atgggtgact ggagcgcctt
aggcaaactc cttgacaagg ttcaagccta ctcaactgct 61 ggagggaagg
tgtggctgtc agtacttttc attttccgaa tcctgctgct ggggacagcg 121
gttgagtcag cctggggaga tgagcagtct gcctttcgtt gtaacactca gcaacctggt
181 tgtgaaaatg tctgctatga caagtctttc ccaatctctc atgtgcgctt
ctgggtcctg 241 cagatcatat ttgtgtctgt acccacactc ttgtacctgg
ctcatgtgtt ctatgtgatg 301 cgaaaggaag agaaactgaa caagaaagag
gaagaactca aggttgccca aactgatggt 361 gtcaatgtgg acatgcactt
gaagcagatt gagataaaga agttcaagta cggtattgaa 421 gagcatggta
aggtgaaaat gcgagggggg ttgctgcgaa cctacatcat cagtatcctc 481
ttcaagtcta tctttgaggt ggccttcttg ctgatccagt ggtacatcta tggattcagc
541 ttgagtgctg tttacacttg caaaagagat ccctgcccac atcaggtgga
ctgtttcctc 601 tctcgcccca cggagaaaac catcttcatc atcttcatgc
tggtggtgtc cttggtgtcc 661 ctggccttga atatcattga actcttctat
gttttcttca agggcgttaa ggatcgggtt 721 aagggaaaga gcgaccctta
ccatgcgacc agtggtgcgc tgagccctgc caaagactgt 781 gggtctcaaa
aatatgctta tttcaatggc tgctcctcac caaccgctcc cctctcgcct 841
atgtctcctc ctgggtacaa gctggttact ggcgacagaa acaattcttc ttgccgcaat
901 tacaacaagc aagcaagtga gcaaaactgg gctaattaca gtgcagaaca
aaatcgaatg 961 gggcaggcgg gaagcaccat ctctaactcc catgcacagc
cttttgattt ccccgatgat 1021 aaccagaatt ctaaaaaact agctgctgga
catgaattac agccactagc cattgtggac 1081 cagcgacctt caagcagagc
cagcagtcgt gccagcagca gacctcggcc tgatgacctg 1141 gagatctag
REFERENCES
Becker, D. L., Evans, W. H. Green, C. R., Warner, A. (1995):
Functional analysis of amino acid sequences in connexin 43 involved
in intercellular communication through gap junctions. J. Cell Sci.
108, 1455 1467. Becker, D. L., McGonnell, I., Makarenkova, H. P.,
Patel, K., Tickle, C., Lorimer, J. and Green, C. R. (1999). Roles
for a1 connexin in morphogenesis of chick embryos revealed using a
novel antisense approach. Devel. Genetics, 24, 33 42. Cotrina, M.
L., Kang, J., Lin, J. H-C., Bueno, E., Hansen, T. W., He, L., Lie,
Y. and Nedergaard, M. (1998). Astrocytic gap junctions remain open
during ischemic conditions. J. Neurosci., 18, 2520 2537. Giaume, C.
and McCarthy, K. D. (1996). Control of gap-junctional communication
in astrocytic networks. TINS, 19, 319 325. Gourdie, R. G., Green,
C. R., Severs, N. J. (1991). Gap junction distribution in adult
mammalian myocardium revealed by an anti-peptide antibody and laser
scanning confocal microscopy. J. Cell Sci. 99: 41 55. Green, C. R.,
Bowles, L., Crawley, A., Tickle C. (1994): Expression of the
connexin 43 gap junctional protein in tissues at the tip of the
chick limb bud is related to epithelial-mesenchymalinteractions
that mediate morphogenesis. Devel. Biol., 161, 12 21. Lin, J. H.
Weigel, H., Cotrina. M. L., Liu, S., Bueno. E., Hansen, A. J.,
Hansen, T. W., Goldman, S. and Nedergaard, M. (1998).
Gap-junction-mediated propagation and amplification of cell injury.
Nature Neurosci. 1, 431 432. Neckers, L., Whitesell. L. (1993):
Anti-sense technology: biological utility and practical
considerations. Am. J. Physiol. 265 (lung cell mol physiol), L1
L12. Simons, M., Edelman, E. R. DeKeyser, J. L., Langer, R.,
Rosenberg, R. D. (1992): Anti-sense c-myb oligonucleotides inhibit
intimal arterial smooth muscle cell accumulation in vivo. Nature,
359, 67 70. Stein, C. A. (1992): Anti-sense
oligodeoxynucleotides--promises and pitfalls, Leukemia 6, 967 974.
Wagner, R. W. (1994): Gene inhibition using anti-sense
oligodeoxynucleotides, Nature, 372, 333 335.
SEQUENCE LISTINGS
1
12130DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 1gtaattgcgg caagaagaat tgtttctgtc
30230DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 2gtaattgcgg caggaggaat tgtttctgtc
30330DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 3ggcaagagac accaaagaca ctaccagcat
30427DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 4tcctgagcaa tacctaacga acaaata 27525DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide
5cgtccgagcc cagaaagatg aggtc 25625DNAArtificial SequenceDescription
of Artificial Sequence Oligonucleotide 6tttcttttct atgtgctgtt ggtga
25730DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 7gacagaaaca attcctcctg ccgcaattac
30830DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 8gtagttacga caggaggaat tgttctcgtc
30930DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 9tcgaactgtc aagactgcta tggcgatcat
301028DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 10ttgtgattta tttagttcgt ctgatttc
281130DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide 11gacagaaaca attcctcctg ccgcaattac 30121149DNAHomo
sapiens 12atgggtgact ggagcgcctt aggcaaactc cttgacaagg ttcaagccta
ctcaactgct 60ggagggaagg tgtggctgtc agtacttttc attttccgaa tcctgctgct
ggggacagcg 120gttgagtcag cctggggaga tgagcagtct gcctttcgtt
gtaacactca gcaacctggt 180tgtgaaaatg tctgctatga caagtctttc
ccaatctctc atgtgcgctt ctgggtcctg 240cagatcatat ttgtgtctgt
acccacactc ttgtacctgg ctcatgtgtt ctatgtgatg 300cgaaaggaag
agaaactgaa caagaaagag gaagaactca aggttgccca aactgatggt
360gtcaatgtgg acatgcactt gaagcagatt gagataaaga agttcaagta
cggtattgaa 420gagcatggta aggtgaaaat gcgagggggg ttgctgcgaa
cctacatcat cagtatcctc 480ttcaagtcta tctttgaggt ggccttcttg
ctgatccagt ggtacatcta tggattcagc 540ttgagtgctg tttacacttg
caaaagagat ccctgcccac atcaggtgga ctgtttcctc 600tctcgcccca
cggagaaaac catcttcatc atcttcatgc tggtggtgtc cttggtgtcc
660ctggccttga atatcattga actcttctat gttttcttca agggcgttaa
ggatcgggtt 720aagggaaaga gcgaccctta ccatgcgacc agtggtgcgc
tgagccctgc caaagactgt 780gggtctcaaa aatatgctta tttcaatggc
tgctcctcac caaccgctcc cctctcgcct 840atgtctcctc ctgggtacaa
gctggttact ggcgacagaa acaattcttc ttgccgcaat 900tacaacaagc
aagcaagtga gcaaaactgg gctaattaca gtgcagaaca aaatcgaatg
960gggcaggcgg gaagcaccat ctctaactcc catgcacagc cttttgattt
ccccgatgat 1020aaccagaatt ctaaaaaact agctgctgga catgaattac
agccactagc cattgtggac 1080cagcgacctt caagcagagc cagcagtcgt
gccagcagca gacctcggcc tgatgacctg 1140gagatctag 1149- -
* * * * *